It is well known that many tumor suppressor genes are methylated in tumor cells. As such, the use of methylation markers has been suggested for the detection or monitoring of cancer in patients. A number of different methods have been proposed for detection of these methylated sequences.
Methylation specific PCR (MSP) is the most commonly used method for detecting methylated or unmethylated DNA. MSP involves the step of bisulfite conversion. Sodium bisulfite is used to deaminate cytosine to uracil while leaving 5-methyl-cytosine intact. Methylation-specific PCR uses PCR primers targeting the bisulfite induced sequence changes to specifically amplify either methylated or unmethylated alleles. Bisulfite conversion destroys about 95% of the DNA. Since DNA concentrations are typically very low in the serum or plasma, a 95% reduction in DNA results in a detection rate of less than 50%.
Alternative methods use restriction enzymes that digest specifically either the methylated or unmethylated DNA. Enzymes that cut specifically methylated DNA are rare. However, enzymes that cut specifically unmethylated DNA are more readily available. Detection methods then establish whether digestion has occurred or not, and thus depending on the specificity of the enzyme used, allows detection of whether the underlying DNA was methylated or unmethylated and thus associated with cancer or not.
Methylation-sensitive enzyme digestion has been previously proposed. For example, Silva et al, British Journal of Cancer, 80:1262-1264, 1999 conducted methylation-sensitive enzyme digestion followed by PCR. However, as noted by the authors Yegnasubramanian et al, Nucleic Acids Research, Vol. 34, No. 3, 2006 e19, such methods are plagued by the number of false-positives that are generated.
The present invention seeks to provide enhanced methods of methylation-sensitive detection which eliminate or reduce false positives and/or false negatives.